Method for Inhibiting Cancer Using Arsenic Trioxide

ABSTRACT

The invention provides a method for treating cancers that are dependent on cyclin D1 for proliferation, survival, metastasis and differentiation, involving administering a composition containing an effective amount of arsenic trioxide to an affected patient. The arsenic trioxide can be administered orally, for example, as a solution, suspension, syrup, emulsion, tablet, or capsule. The composition can also contain one or more pharmaceutically acceptable carriers and/or excipients.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. Ser. No. 10/669,869filed Sep. 23, 2003, which claims priority to U.S. Ser. No. 60/417,200filed Oct. 9, 2002 and U.S. Ser. No. 60/483,014 filed Jun. 25, 2003, andis also a continuation-in-part of 11/549,347 filed Oct. 13, 2006 and allof which are incorporated by reference in their entirety.

FIELD OF INVENTION

This invention relates to methods of inhibiting cancer by affectingexpression, translation, and biological activity of cancersoverexpressing or dependent on cyclin D1 using arsenic trioxide.

BACKGROUND OF THE INVENTION

Mantle cell lymphoma (MCL) is a well-defined subtype of B cell lymphomain the World Health Organization classification, and accounts forapproximately 3-10% of all non-Hodgkin lymphomas. The chromosomalaberration t(11,14)(q13;q32) can be found in practically all cases ofMCL. The translocation results in juxtaposition of the immunoglobulinheavy chain joining region on chromosome 14 to the cyclin D1 gene onchromosome 11. The molecular consequence of the translocation is toplace cyclin D1 under the control of the immunoglobulin heavy chain geneenhancer, leading to over-expression of the cyclin D1 protein.

Although MCL accounts for approximately 3-8% of B-cell lymphomas, it isdifficult to manage. Initial treatment with rituximab plus combinationchemotherapy or purine analogues results in complete remission (CR)rates varying from 34-87%. However, relapses occur in most patients withprolonged follow up. Treatment options for relapsed patients arelimited. Several approaches have been adopted, including the use of theproteasome inhibitor bortezomib, thalidomide and the mammalian target ofrapamycin (mTOR) inhibitor temsirolimus. The overall response (OR) ratesof these agents varied from 38-81%, but the CR rate was only 3-31%.Therefore, there is an urgent need to define effective treatmentstrategies for MCL.

It is an object of this invention to provide agents and methods fortreating cancers such as MCL and other cancers over-expressing cyclinD1.

It is another object of this invention to provide methods, strategies,doses, and dosing schedules for the administration of As₂O₃ in theclinical inhibition of cancers over-expressing cyclin D1.

SUMMARY OF THE INVENTION

It has been discovered that As₂O₃ suppresses cyclin D1 and initiatesdown-regulation of cyclin D1 by activating GSK-3 β, which phosphorylatescyclin D1. Activation of IKKb leads to phosphorylation of cyclin D1,which is ubiquitinated. Ubiquitinated cyclin D1 is degraded in theproteasome. This is the basis for the discovery that MCL and othercancers over-expressing cyclin D1 can be treated with As₂O₃, preferablyoral As₂O₃.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a line graph showing As₂O₃ (concentration in micromolar)percent induced apoptosis in MCL cells, based on a MTT test of Jeko-1and Granta-519 cells treated for 72 hours with As₂O₃. There was a doseand time dependent suppression of cellular proliferation. Viabilitysignificantly decreased at or above 1 μM As₂O₃ as compared with baseline(one-way ANOVA with Dunnett's post-tests, p<0.05) (triplicateexperiments)

FIG. 1B is a scatter plot of popidium iodide versus annexin expressionin cells treated with As₂O₃. There was a significant increase inapoptotic cells after As₂O₃ treatment. (#: apoptotic cells that wereannexin V positive and popidium iodide negative).

FIGS. 2A and 2B show down-regulation of cyclin D1 by As₂O₃ treatment.FIG. 2A: As₂O₃ (4 μM) induced a time dependent down-regulation of cyclinD1 in Jeko-1 and Granta-519 cells. Triplicate experiments and arepresentative Western blot demonstrate significant decrease in cyclinD1 level after 2 hours (one-way ANOVA with Dunnett's post-tests,p<0.05). FIG. 2B: As₂O₃ (treatment for 8 hours) induced a dose dependentdown-regulation of cyclin D1 in Jeko-1 and Granta-519 cells. Triplicateexperiments demonstrate significant decrease in cyclin D1 level at orabove 2 μM (one-way ANOVA with Dunnett's post-tests, p<0.05).

FIG. 3 shows dephosphorylation of retinoblastoma (RB) by As₂O₃ treatmentin MCL lines. As₂O₃ treatment resulted in dephosphorylation of RB(significant decrease of phosphor-Rb Ser-795 at or more that 8 hours ofAs₂O₃ treatment, triplicate experiments, one-way ANOVA with Dunnett'spost-tests, p<0.05).

FIGS. 4A, 4B and 4C show As₂O₃ treatment induced phosphorylation ofcyclin D1 and GSK-3. FIG. 4A. Cell lysates immunoblotted withanti-phospho-cyclin D1 (Thr-286). As₂O₃ treatment led to significantlyincreased phosphor-cyclin D1 (triplicate experiments, one-way ANOVA withDunnett's post-tests, p<0.05). FIG. 4B. Cell lysates immunoblotted withanti-phospho-cyclin GSK-3,p (Try-216). As₂O₃ treatment led tosignificantly increased phosphor-GSK-3β (triplicate experiments, one-wayANOVA with Dunnett's post-tests, p<0.05). FIG. 4C. Pre-incubation with6-bromoindirubin-3′-oxime (BIO; 10 μM) before As₂O₃ treatment (4 μM, 8hour, 37° C.) prevented cyclin D1 down-regulation, showing that GSK-3bwas involved. Result a significant reduction of cyclin D1 as comparedwith control (triplicate experiments, one-way ANOVA with Dunnett'spost-tests, p<0.05).

FIGS. 5A and 5B show that IKK was involved in As₂O₃-induceddown-regulation of cyclin D1. FIG. 5A. As₂O₃ treatment (4 μM for 2hours) led to a significant increase in phosphor-IKKα/β (Ser-176/180)(triplicate experiments, one-way ANOVA with Dunnett's post-tests,p<0.05). FIG. 5B. Pre-incubation with the IKK inhibitor BMS (10 mM, 30minutes) successfully prevented As₂O₃-induced cyclin D1 down-regulation(triplicate experiments, one-way ANOVA with Dunnett's post-tests,p<0.05).

FIG. 6 shows As₂O₃-induced ubiquitination of cyclin D1 in MCL. Celllysates were immunoprecipitation with anti ubiquitin (Ub) or anti-cyclinD1 antibody. The immunoprecipitates and the crude lysates wereimmunoblotted with anti-cyclin D1 and anti-ubiquitin antisera. As₂O₃induced a significant increase in binding between cyclin D1 andubiquitin (increase in ubiquitination from 30 minutes to 2 hours afterAs₂O₃ treatment as compared to the baseline, triplicate experiments,one-way ANOVA with Dunnett's post-tests, p<0.05).

FIGS. 7A and 713 show As₂O₃-induced cyclin D1 degradation involved theproteasome but not the lysosome in MCL. FIG. 7A. Pre-incubation with theproteasome inhibitors MG132 (MG, 30 μM), bortezomib (bort, 10 μg/ml) andlactacystin (lact, 10 μM) successfully prevented As₂O₃ induced cyclin D1degradation. FIG. 7B. Pre-incubation with the lysosomal inhibitorammonium chloride (NH₄Cl, 2.5 mM) was ineffective in preventingAs₂O₃-induced cyclin D1 degradation.

FIG. 8 is a schematic diagram showing the proposed mechanism ofdegradation of cyclin D1 mediated by As₂O₃.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Arsenic Trioxide Formulations

Arsenic Trioxide

Arsenic trioxide is available from a number of different suppliers.Arsenic trioxide is an amphoteric oxide which is known for its acidicproperties. It dissolves readily in alkaline solutions to givearsenites. It is much less soluble in acids, but will dissolve inhydrochloric acid to give arsenic trichloride or related species. Itreacts with oxidizing agents such as ozone, hydrogen peroxide and nitricacid to give arsenic pentoxide, As₂O₅. It is also readily reduced toarsenic, and arsine (AsH₃) may also be formed.

Arsenic trioxide has many uses including as: a starting material forarsenic-based pesticides; a starting material for arsenic-basedpharmaceuticals, such as a neosalvarsan, a synthetic organoarsenicantibiotic; a decolorizing agent for glasses and enamels, a woodpreservative, and a cytostatic in the treatment of refractorypromyelocytic (M3) subtype of acute myeloid leukemia.

An oral arsenic trioxide (As₂O₃) is highly efficacious for relapsedacute promyelocytic leukemia. Oral As₂O₃ causes a smaller prolongationof QT intervals, and therefore is a much safer drug for treatingleukemia.

Formulations

The following delivery systems, which employ a number of routinely usedpharmaceutical carriers, are only representative of the many embodimentsenvisioned for administering the instant compositions.

Parenteral Formulations

Injectable drug delivery systems include solutions, suspensions, gels,microspheres and polymeric injectables, and can comprise excipients suchas solubility-altering agents (e.g., ethanol, propylene glycol andsucrose) and polymers (e.g., polycaprylactones and PLGA's). Implantablesystems include rods and discs, and can contain excipients such as PLGAand polycaprylactone.

Enteral Formulations

Oral delivery systems include solid dosage forms such as tablets (e.g,compressed tablets, sugar-coated tablets, film-coated tablets, andenteric coated tablets), capsules (e.g., hard or soft gelatin ornon-gelatin capsules), blisters, and cachets. These can containexcipients such as binders (e.g., hydroxypropylmethylcellulose,polyvinyl pyrilodone, other cellulosic materials and starch), diluents(e.g., lactose and other sugars, starch, dicalcium phosphate andcellulosic materials), disintegrating agents (e.g., starch polymers andcellulosic materials) and lubricating agents (e.g., stearates and talc).The solid dosage forms can be coated using coatings and techniques wellknown in the art.

Oral liquid dosage forms include solutions, syrups, suspensions,emulsions, elixirs (e.g., hydroalcoholic solutions), and powders forreconstitutable delivery systems. The formulations can contain one ormore carriers or excipients, such as suspending agents (e.g., gums,zanthans, cellulosics and sugars), humectants (e.g., sorbitol),solubilizers (e.g., ethanol, water, PEG, glycerin, and propyleneglycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens, andcetyl pyridine), emulsifiers, preservatives and antioxidants (e.g.,parabens, vitamins E and C, and ascorbic acid), anti-caking agents,coating agents, chelating agents (e.g., EDTA), flavorants, colorants,and combinations thereof. The compositions can be formulated as a foodor beverage (e.g., a shake) containing buffer salts, flavoring agents,coloring agents, sweetening agents, and combinations thereof.

Topical Formulations

Transmucosal delivery systems include patches, tablets, suppositories,pessaries, gels and creams, and can contain excipients such assolubilizers and enhancers (e.g., propylene glycol, bile salts and aminoacids), and other vehicles (e.g., polyethylene glycol, fatty acid estersand derivatives, and hydrophilic polymers such ashydroxypropylmethylcellulose and hyaluronic acid).

Dermal delivery systems include, for example, aqueous and nonaqueousgels, creams, multiple emulsions, microemulsions, liposomes, ointments,aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon basesand powders, and can contain excipients such as solubilizers, permeationenhancers (e.g., fatty acids, fatty acid esters, fatty alcohols andamino acids), and hydrophilic polymers (e.g., polycarbophil andpolyvinylpyrolidone). In one embodiment, the pharmaceutically acceptablecarrier is a liposome or a transdermal enhancer.

II. Methods of Treatment

Cyclin D1 is a D-type cyclin critically involved in the control of thecell cycle. It assembles with its catalytic partners cyclin-dependentkinase 4 (CDK4) and CDK6 to form an active holoenzyme complex, whichcontrols G1 progression and G1/S transition. The active holoenzymecomplex phosphorylates the retinoblastoma protein RB. Phosphorylated RBreleases the E2F family of transcription factors from inhibition,enabling E2Fs to coordinately regulate genes necessary for DNAreplication and hence progression into S phase. Over-expression ofcyclin D1 is demonstrable in many cancers, including cancers of thedigestive tract, cancers of the female genital tract, and malignantlymphomas.

Owing to its important influence on the cell cycle, cyclin D1 expressionis carefully regulated. Cyclin D1 gene mRNA and transcription appears tobe constant through the cell cycle. However, a decline in cyclin D1level occurs during S phase, which has been attributed to its increasedproteasomal degradation. Cyclin D0 phosphorylation at a threonineresidue 286 (Thr-286) positively regulates its proteasomal degradation.Thr-286 phosphorylation is mediated by glycogen synthase kinase-3β(GSK-3β). In addition to targeting cyclin D1 to proteosomes,GSK-3β-induced Thr-286 phosphorylation also promotes cyclin D1 nuclearexport, by increasing the binding of cyclin D1 to a nuclear exportinCRM1. IkappaB kinase (IKK) alpha, IKKα, associates with andphosphorylates cyclin D1 also at Thr-286, thereby participating in thesubcellular localization and turnover of cyclin D1.

As₂O₃ induced apoptosis in MCL lines at 2-4 μM, which is within theplasma levels achieved after As₂O₃ therapy. As₂O₃ induces a dose andtime dependent suppression of cyclin D1. The suppression of cyclin D1restores RB to a hypophosphorylated state, in parallel with a change incell cycle. These biologic changes are consistent with the apoptosisobserved upon As₂O₃ treatment.

The down-regulation of cyclin D1 mediated by As₂O₃ occurs at apost-transcriptional level since cyclin D1 is under the transcriptionalcontrol of the immunoglobulin heavy chain gene enhancer in MCL, which isunlikely to be affected by As₂O₃. Furthermore, in physiologicconditions, the control of cyclin D1 during the cell cycle is alsomediated in part via alteration in the stability of cyclin D1. Thisprocess is controlled by phosphorylation of cyclin D1 at Thr-286, aprocess mediated by GSK-3β. GSK-3β is itself tightly regulated. Mitogensinactivate GSK-3β by a pathway involving Ras, phosphatidylinositol 3kinase (PI3K), and protein kinaseB/Akt. Ras activates PI3K, which inturn activates Akt. Akt inactivates GSK-3β by phosphorylating it atserine residue 9. This removes the inhibition of GSK-3β on cyclin D1,allowing cyclin D1 to accumulate and thus activate cell cycling. GSK-3βcan also be activated by phosphorylation at a tyrosine residue 216(Try-216) in the kinase domain. As₂O₃-mediates an increase of GSK-30Try-216 phosphorylation. The end result of As₂O₃-mediated increase inGSK-30 Try-216 phosphorylation is the increase in cyclin D1 Thr-286phosphorylation, a key step in its degradation.

The IKK complex is the major regulatory component in the NK-κB pathway.It comprises the catalytic subunits IKKα and IKKβ, and a regulatorysubunit IKKγ/NEMO. IKKα has been shown to phosphorylate cyclin D1 atThr-286, the same site targeted by GSK-3b. IKKα needs to be activated byphosphorylation at a serine residue 176 (Ser-176) before participatingin the regulation of NF-κB by phosphorylating IκB. IKKα Ser-176phosphorylation is mediated by NK-κB inducing kinase (NIK).As₂O₃-induces an increase in IKK phosphorylation. As₂O₃-mediates anincrease in physical interaction between IKK and cyclin D1, as shown inimmunoprecipitation experiments. An IKK specific inhibitor BMS-345541alleviated As₂O₃-induced cyclin D1 down-regulation. These resultsindicate that IKK is also an effector of As₂O₃ treatment.

As₂O₃-mediated cyclin D1 Thr-286 phosphorylation increases itsubiquitination. The time course of ubiquitination is commensurate withthe timing of the biologic functions of As₂O₃ on the MCL lines. AfterAs₂O₃ treatment, increased ubiquitination is first detected at 30minutes and continues to increase. At two hours, significantdown-regulation of cyclin D1 is first observed, which is associated witha parallel hypophosphorylation of RB. Significant activation of caspase3 is observed at four hours. These sequence of events are consistentwith cyclin D1 down-regulation initiated by Thr-286 phosphorylation.

Cyclin D1 is a cytosolic and nuclear protein. Therefore,polyubiquitination is involved, which targets the protein to degrade inproteasomes. Inhibition of proteasomes successfully preventedAs₂O₃-induced down-regulation of cyclin D1. Inhibition of lysosomes, thesite of degradation of monoubiquitinated proteins, does not interferewith As₂O₃-induced down-regulation of cyclin D1. These results confirmthat As₂O₃ down-regulated cyclin D1 by promoting its proteasomaldegradation.

Arsenic trioxide can be used for the treatment of cancers that aredependent on cyclin D1 for proliferation, survival, metastasis anddifferentiation.

Patients with cancers that overexpress cyclin D can be treated withAs₂O₃, Mantle cell lymphoma is a cancer characterized by overexpressionof cyclin D, as are cancers of the digestive tract, cancers of thefemale genital tract, and malignant lymphomas.

The dose of oral As₂O₃ is typically adjusted according to age and kidneyfunction. In one embodiment, the dose range of As₂O₃ varies from 1 to 10mg, typically about 5 to 10 mg.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLES Example 1 In Vitro Studies Show As₂O₃ is Effective in Treatmentof MCL by Targeting Cyclin D1

Materials and Methods

Cell lines. The MCL lines Jeko-1 and Granta-519 were obtained fromGerman Collection of Microorganisms and Cell Cultures (ACC 553 and ACC342, Braunschweig, Germany). Jeko-1 cells were cultured in RPMI 1640with 20% fetal bovine serum (FBS), and Granta-519 cells in DMEM with 10%FBS; both with 50 units/ml penicillin and 50 μg/ml streptomycin, at 5%CO₂.

Reagents and antibodies. Reagents and antibodies used included cellculture reagents (Invitrogen, Carlsbad, Calif., USA); kinase inhibitorsand their inactive analogues (Calbiochem, Darmstadt, Germany); antiserumto phospho-GSK3 (tyrosine 216, Try-216) (Upstate, Lake Placid, N.Y.,USA); antisera to cyclin D1, phospho-cyclin D1 (Thr-286), GSK3β,phospho-GSK3β (Tyr-216), IκB kinase (IKK)α/β, phospho-IKKα/β (serine1761180, Ser-176/180), RB and phospho-RB (serine 795, Ser-795),caspase-3 and β-actin (Cell Signaling Technology, Beverly, Mass., USA);protein G-agarose (Upstate); ECL kit (Amersham, Piscataway, N.J., USA);cell proliferation kit I (MTT) (Roche Applied Science, Indianapolis,Ind., USA); annexin V-FITC Kit (Beckman Coulter, Fullerton, Calif.,USA); and RNeasy Kit and One-Step RT-PCR Kit (Qiagen, Valencia, Calif.,USA).

Cell viability assays. Cells were seeded on 96-well microplates at2×10⁴/well in 100 ml growth medium containing different concentration ofAs₂O₃ as indicated at 37° C. for 72 hours. MTT labeling reagent (10 μl,5 mg/ml) (Roche Applied Science, Indianapolis, Ind., USA) was added toeach well at 37° C. for 4 hours, followed by 100 μl solubilization at37° C. overnight. Solubilized fomarzan crystals were quantifiedspectophotometrically at 590 nm with a microplate ELISA reader.

Apoptosis assay. Cells were seeded at 1×10⁶/ml in differentconcentrations of As₂O₃ as indicated at 37° C. for 24 hours, harvested,rinsed in ice-cold phosphate buffered saline (PBS), and resuspended in500 μl binding buffer containing annexin V-FITC and propidium iodide(PI) (Beckman Coulter, Fullerton, Calif., USA) for 20 minutes on ice.The percentages of apoptotic cells (annexin-V positive, PI negative)were determined on a flow cytometer (Epics, Beckman Coulter) withappropriate color compensation.

Cell Cycle Analysis. Cells were seeded at 1×10⁵/ml in differentconcentrations of As₂O₃ as indicated at 37° C. for 8 hours, harvested,washed in ice-cold PBS, resuspended in 500 μl PBS, stained with PI for10 minutes on ice. Cell cycle was determined by flow cytometry (Epics,Beckman Coulter).

Semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) for cyclin D1. Cells were seeded at a density of 1×10⁶/ml indifferent concentrations of As₂O₃ at 37° C. for 8 hours, washed with PBSbuffer and lysed with RTL buffer. RNA was extracted with an RNeasy Kit,followed by cDNA synthesis and a 30-cycle PCR with a One-Step RT-PCR Kitwith the forward primer 5′-CTG GCC ATG AAC TAC CTG GA-3′ and the reverseprimer 5′-GTC ACA CTT GAT CAC TCT GG-3′. Cycling conditions weredenaturation (1 minute at 94° C., first cycle 5 minutes), annealing (2minutes at 50° C.) and extension (3 minutes at 72° C., last cycle 10minutes).

Western Blotting Analysis. Cells were seeded at a density of 1×10⁶/mlovernight. Where applicable, cells were pre-treated with variousinhibitors for 30 minutes, and then incubated with 4 μM As₂O₃ fordifferent time periods as indicated. Cells were lysed in lysis buffer(50 mM Tris-HCl, 100 mM NaCl, 5 mM EDTA, 40 mM NaP₂O₇, pH 7.5, 1% TritonX-100, 4 μg/ml aprotinin, 1 mM dithiothreitol, 200 μM Na₃VO₄, 0.7 μg/mlpepstatin, 100 μM phenylmethylsulfonyl fluoride, and 2 μg/ml leupeptin).Clarified lysates were resolved on 12% SDS-phenylmethylsulfonyl fluorideand transferred to nitrocellulose membranes. The membranes were blockedwith 5% non-fat milk, washed, incubated with the appropriate antibodiesfollowed by horseradish peroxidase-conjugated secondary antisera.Immuno-reactive bands were visualized by chemiluminescence with the ECLkit, detected on X-ray films and quantified by densitometric scanning(Eagle Eye II still video system, Stratagene, La Jolla, Calif., USA).

Coimmunoprecipitation Assays. Cells were seeded at 1×10⁶/ml overnight,treated with 4 μM As₂O₃ at 37° C. for different time periods asindicated, and lysed in lysis buffer, Cell lysates were incubated withan anti-cyclin D1, anti-ubiquitin, anti-calpain 2 or anti-IKKα/βantibodies (4 μg/sample) at 4° C. for 1 hour, followed by incubationwith 30 μl of protein G-agarose (50% slurry) at 4° C. for another 2hour. Immunoprecipitates were washed four times with 400 μl lysisbuffer, resuspended in 50 μl lysis buffer and 10 ml 6× sample buffer andboiled for 5 minutes. Immunoprecipitates were then analysed by Westernblot analysis.

Results

As₂O₃ induced dose and time dependent apoptosis in MCL cells.

The MTT test showed that As₂O₃ induced a dose-dependent cytotoxicity inJeko-1 and Granta-519 cells. Flow cytometric analysis showed that As₂O₃treatment led to induction of apoptosis. Western blot analysis showedthat caspase 3 activation was involved in As₂O₃-induced apoptosis.

FIGS. 1A and 1B are graphs showing As₂O₃ (concentration in microM)percent induced apoptosis in MCL cells measured using a MTT test ofJeko-1 and Cranta-519 cells treated for 72 hours with As₂O₃. There was adose and time dependent suppression of cellular proliferation. Viabilitysignificantly decreased at or above 1 μM As₂O₃ as compared with baseline(one-way ANOVA with Dunnett's post-tests, p<0.05) (triplicateexperiments). (. Significant increase in apoptotic cells after As₂O₃treatment. #: apoptotic cells that were annexin V positive and popidiumiodide negative). Western Blotting showed activation of caspase 3 byAs₂O₃ treatment, 0, 1.5 and 2.5 microM. Cleaved caspase 3 weredetectable four hours after As₂O₃ treatment.

Cyclin D1 was down-regulated in MCL by As₂O₃. To determine the molecularmechanisms of As₂O₃-induced apoptosis in MCL, the expression of cyclinD1 was examined. Western blot analysis showed that As₂O₃-induced a timeand dose dependent suppression of cyclin D1 in both Jeko-1 andGranta-519 cell lines. Treatment with As₂O₃ at 4 μM led to suppressionof cyclin D1, first detectable at 2 hours and almost complete at 8-12hours As₂O₃ suppression of cyclin D1 was also dose-dependent. Triplicateexperiments demonstrate significant decrease in cyclin D1 level after 2hours (one-way ANOVA with Dunnett's post-tests, p<0.05) Triplicateexperiments demonstrate significant decrease in cyclin D1 level at orabove 2 μM (one-way ANOVA with Dunnett's post-tests, p<0.05).Semi-quantitative polymerase chain reaction showing that cyclin D1 genetranscription was unaffected by As₂O₃ treatment.

As₂O₃ induced down-regulation of cyclin D1 disrupted its signaling. Toinvestigate if cyclin D1 down-regulation is biologically relevant, RBphosphorylation was investigated. As₂O₃ treatment led to a timedependent decrease in RB phosphorylation, which occurred at a similartime-frame as compared with cyclin D1 down-regulation. Cell cycleanalysis by flow cytometry showed that there was an increase in theproportion of apoptotic cells.

Down-regulation of cyclin D1 by As₂O₃ was post-transcriptional. RT-PCRshowed that cyclin-D1 gene transcription was unaffected by As₂O₃treatment of up to 8 μM, suggesting that the down-regulation of cyclinD1 was post-transcriptional.

As₂O₃-induced cyclin D1 down-regulation was related to GSK3β activation.Western blot analysis showed that As₂O₃ treatment resulted insignificant increases in cyclin D1 phosphorylation at Thr-286, aprerequisite for cyclin D1 degradation. Cyclin D1 phosphorylation byGSK-3β requires prior activation of GSK-3β by phosphorylation atTyr-216. As₂O₃ treatment significantly increased GSK-3β Tyr-216phosphorylation, indicating that GSK-3β might mediate As₂O₃-inducedcyclin D1 phosphorylation and hence degradation. To confirm the role ofGSK-3β as a mediator of As₂O₃, Jeko-1 cells were pre-incubated with theGSK-3β inhibitor 6-bromoindirubin-3′-oxime (BIO; 10 μM) before As₂O₃treatment. The results showed that BIO successfully preventedAs₂O₃-induced down-regulation of cyclin D1. Collectively, theseobservations indicate that As₂O₃ down-regulated cyclin D1post-transcriptionally, probably by increasing its degradation.

As₂O₃-induced cyclin D1 down-regulation was also dependent on IKKα/β. Todetermine if IKK was involved in As₂O₃-induced down-regulation of cyclinD1, IKKα/β phosphorylation at Ser-178/180 was examined. As₂O₃significantly increased IKKα/β Ser-178/180 phosphorylation, which wasrequired for activation of IKKα/β (FIG. 5A). Pre-treatment with theIKKα/β inhibitor BMS-345541 (BMS; 10 μM) significantly preventedAs₂O₃-induced cyclin D1 down-regulation, suggesting that IKKα/β was amolecular mediator of As₂O₃ (FIG. 5B). Immunoprecipitation with ananti-IKKα/β antibody showed that cyclin D1 bound IKKα/β. Similarly, whencyclin D1 was immunoprecipitated, IKKα/β was also confirmed toco-immunoprecipitate. These results confirmed that As₂O₃ activatedIKKα/β, which participated in the down-regulation of cyclin D1.

As₂O₃ promoted cyclin D1 ubiquitination. To study if As₂O₃-inducedcyclin D1 down-regulation was mediated via ubiquitination,immunoprecipitation experiments were performed on lysates from Jeko-1cells treated with As₂O₃. Immunoprecipitation with an anti-ubiquitinantibody showed a time-dependent increase in bound cyclin D1 (FIGS. 6Aand B). Similarly, lysates immunoprecipitated with an anti-cyclin D1antibody also showed a time dependent increase in bound ubiquitin. Theseresults showed that As₂O₃ promoted cyclin D1 ubiquitination, confirmingthat As₂O₃-induced GSK-31 and IKKα/β activation was biologicallyrelevant.

As₂O₃ induced cyclin D1 degradation in 26S and 20S proteasomes but notlysosomes. Pre-incubation of Jeko-1 cells with the 26S and 20Sproteosome inhibitors MG132 (30 μM), bortezimab (10 μg/ml) andlactacystin (10 μM) attenuated As₂O₃-induced cyclin D1 down-regulation(FIG. 7A). However, pre-incubation with the lysosomal inhibitor ammoniumchloride (NH₄Cl) had no effect on As₂O₃-induced down-regulation ofcyclin D1 (FIG. 7B). The results confirmed that As₂O₃ down-regulatedcyclin D1 by promoting its ubiquitination, hence targeting it to theproteosome for degradation.

Overall model. An overall model of the action of As₂O₃ on MCL is shownin FIG. 8.

Example 2 Clinical Study of Oral-As₂O₃ in the Treatment of Patients withRefractory and Relapsed MCL that Over-Expressed Cyclin D1

Materials and Methods

Patients. Consenting patients with relapsed or refractory B-celllymphomas, and an ECOG performance status of <2 were recruited. Allpatients gave informed consent, and the treatment was approved by theinstitute review board of Queen Mary Hospital.

Treatment. Treatment was initiated with oral-As₂O₃ (10 mg/day forpatients below 70 years old with normal renal function; 5 mg/day forpatients over 70 years old, or with impaired renal function), ascorbicacid (AA, 1 g/day) and chlorambucil (4 mg/day) as outpatients untildisease response or progression was documented. In patients with bulkydisease, debulking with VPP (vincristine 2 mg/day×1, prednisolone 30mg/day×14 and procarabzine 50-100 mg/day×14) was used. After maximumresponse was achieved, chlorambucil was taken off and a maintenanceregimen of As₂O₃ (5-10 mg/day) and AA (1 g/day) was given for two weeksevery 2 months for a planned two years. Responses were classifiedaccording to standard NCI criteria, and monitored by regular physicalexamination, marrow and blood assessment, and computerized tomographicscans.

Results

Characteristics of patients with MCL. Table 1 shows the results of theclinical use of oral-As₂O₃ in patients with refractory or relapsedmantle cell lymphoma that over-expressed cyclin D1. The results showedan overall response rate of 64%. Four patients achieved completeremission (CR), whereas two patients achieved complete remissionunconfirmed. Of the fourteen patients treated (Table 1), eleven hadadvanced relapses (R) (R2, n=5; R3, n=4; R4, n=2). Three patientstreated in R1 had advanced age (76, 77 and 90 years). All but twopatients had received an anthracycline based multi-agent chemotherapy.Other previous treatment included rituximab (n=8), autologoushematopoietic stem cell transplantation (HSCT) (n7-3), and bortezomib(n=1). Other poor prognostic indicators included marrow infiltration(n=11) and extensive extranodal involvement (n=9), so that 12/14 (86%)cases had stage IV disease. The median time from initial diagnosis toAs₂O₃ treatment was 33 (8-85) months. TABLE 1 Clincopathologic featuresand treatment outcome of 14 patients with relapsed or refractory MCLInitial disease Current relapse Total Outcome and stage sites Previoustreatment Time* No Sites As₂O₃ response survival 1 M/69 III Colon,abdomen FND × 6, COPP × 6 56 m 2 Cervical 140 mg CR off Rx, 28 m+. 2M/63 IV BM, R-CEOP × 6, IMVP × 6 11 m 2 BM, cervical 160 mg CR on Rx, 13m+. generalized LN 3 M/65 IV BM, mesentery, FND × 7, IMVP × 2, 85 m 3Eye 120 mg CR on Rx, 17 m+. generalized LN R-DHAP × 8 4 F/77 IV Pleura,Clb 33 m 1 Groin, jaw 140 mg CR R2 at 16 m, CR generalized LN again withAs₂O₃ + Clb 5 M/70 III Generalized LN COPP × 2, IMVP × 6, 85 m 4Cervical, abdomen 250 mg CRu R5 at 20 m, on Clb As₂O₃ + Clb 6 M/76 IVBM, CEOP × 7 19 m 1 BM, leukemic, eyes,  210 mg* CRu on Rx, 8 m+generalized LN generalized LN 7 M/58 IV BM, CEOP × 6, R-ESHAP × 6 18 m 2Generalized skin 140 mg PR On Rx, 3 m+ generalized LN 8 M/81 IV BM,leukemic, CHOP × 6, ChlVPP × 2 18 m 2 BM, LN, liver,  300 mg* PR died at16 m liver, spleen spleen, leukemic 9 M/51 IV Generalized LN, CVAD × 7,CEOP × 2, 25 m 4 BM, LN, scalp  NA* Static on Rx, 8 m+ spleen, BM,R-DHAP × 3, Thal scalp, eye 10 F/76 IV General LN, BM, R-COPP × 6 12 m 2BM, LN  160 mg* PR died at 6 m scalp 11 M/90 IV BM, leukemic Clb  8 m 1BM, leukemic NA NR died at 4 m 12 M/54 IV Generalized LN, CEOP × 6, DHAP× 1, 36 m 3 BM, generalized LN, NA Static died at 17 m BM, gut, liver,NOPP × 5, Clb spleen spleen, leukemic 13 F/57 IV Generalized LN, CEOP ×6, DHAP × 4, 36 m 3 BM, LN NA NR died at 1 m BM, spleen R-BVP × 3 14M/63 IV Generalized LN, CEOP × 6, AHSCT, 72 m 3 BM, generalized LN NA NRdied at 1 m pleura, BM R-DHAP × 6, Thal, velcade, FNDM: male; F: female; LN: lymphadenopathy; BM: bone marrow; m: months; R:rituximab; CEOP: cyclophospamide, epirubicin, vincristine, prednioloneFND: fludarabine, mitoxantrone, dexamethasone; DHAP: cisplatinum,cytosine arabinoside, dexamethasone; Thal: thalidomideChlvPP: chlorambucil, vincristine, procarbazine, prednisolone; COPP:cyclophosphamide, vincristine, procarbazine, prednisolone; NOPP:mitoxantrone; vincristine, procarbazine, prednisolone; BVP; bleomycin,vinblastine, prednisolone; AHSCT: autologous hematopoietic stem celltransplantationClb: chlorambucil; NA: not available; CR: complete remission; CRu:complete remission (unconfirmed); PR: partial remission; NR; no response

Treatment response. Nine patients responded, giving an OR rate of 64%.Four patients (cases 1-4) achieved CR. Two patients (cases 5, 6)achieved unconfirmed CR (CRu). They had become asymptomatic without anydetectable superficial diseases. Marrow and peripheral blood involvementwas also cleared. However, small residual internal lymph nodes remained.These lymph nodes were negative on gallium scan and had remained staticin size. Three patients had partial responses (PR) with >50% reductionin the size of assessable lymph nodes.

Case 6 had bilateral orbital infiltration at relapse that completelyresolved after 4 months of oral As₂O₃ treatment and ascorbic acid. Case8 who was relapsing in leukemic phase with massive splengomegaly showedpartial remission after 8 months of treatment with oral As₂O₃ andascorbic acid as determined by MRI scans. Histological analysis revealedthat case X had dense marrow infiltration that resolved after 8 monthsof treatment with oral-As₂O₃ and ascorbic acid.

Outcome. Of the four patients with CR, one had relapsed at 16 months.She achieved a CR3 again with daily As₂O₃ and resumption ofchlorambucil. Two patients were still on maintenance As₂O₃+AA treatment,while one had completed the planned two years of treatment. Of the twopatients with CRu, one patient had relapsed at 20 months. He achievedCR5 again with As₂O₃ and chlorambucil therapy. For the three patientswith PR, one patient developed progressive disease while on maintenancetherapy 12 months later and died of refractory lymphoma. Two defaultedtreatment and both relapsed.

Toxicity. Significant (W.H.O grade 3-4) neutropenia and thrombocytopeniawas observed in 7 patients. These patients had previously receivedmultiple chemotherapy, or autologous HSCT. The neutropenia responded tohematopoietic growth factors. No significant sepsis or bleeding wereobserved. Other side effects included fever (n=7), herpes zosterreactivation (n=3), fluid accumulation (n=2), nausea (n=3) and headache(n=2). No significant QT prolongation or arrhythmia was observed. Fivepatients did not report any side effects at all.

AS₂O₃ suppresses MCL cell growth by targeting cyclin D1. AS₂O₃ inducesthe phosphorylation of GSK-3 p and IKK. Cyclin D1 over-expression ispathogenetically important in a vast diversity of cancers. Oral-As₂O₃inhibited refractory or relapsed MCL in 14 patients, whichover-expressed cyclin D1, with an overall response in 9 patients (64%).Four patients achieved complete remission, two patients completeremission unconfirmed, and three patients with partial remissions. Theseresults were very good, given that these patients had refractory orrelapsed disease.

Taken together, the evidence demonstrates that As₂O₃ decreases cyclin D1and that the decrease in cyclin D1 was post-transcriptional. As₂O₃induces GSK-3β and IKK activation and hence phosphorylation of cyclinD1. Phosphorylated cyclin D1 is degraded in the proteasome. Oral As₂O₃induces a high response rate clinically in patients with refractory orrelapsed MCL, a cancer that over-expresses cyclin D1.

1. A method for inhibiting cyclin D1 production in a cell, comprisingcontacting the cell with an amount of arsenic trioxide effective toinhibit cyclin D1 production therein.
 2. The method according to claim1, wherein the cell is a cancer cell.
 3. The method according to claim2, wherein the cell is from a cancer of the digestive tract, cancer ofthe female genital tract, and malignant lymphomas.
 4. The methodaccording to claim 2, wherein the cancer cells are in a patient and thearsenic trioxide is administered orally.
 5. The method according toclaim 4, wherein the cancer cell is from a human female genital tractcancer, a digestive tract cancer, or a malignant lymphoma.
 6. The methodaccording to claim 4 wherein the cell is a human mantle cell lymphoma(MCL).
 7. A unit dosage form for oral administration comprising arsenictrioxide in a pharmaceutically acceptable carrier for enteraladministration.
 8. The unit dosage form of claim 7, further comprisingone or more pharmaceutically acceptable excipients.
 9. The unit dosageform of claim 7, wherein the arsenic trioxide is present in an amountfrom 5 to 10 mg.
 10. The unit dosage form of claim 7, wherein the unitdosage form is selected from the group consisting of solutions,suspensions, emulsions, syrups, tablets, and capsules.
 11. A unit dosageform for oral administration comprising arsenic trioxide in apharmaceutically acceptable carrier for enteral administration, whereinthe dosage form contains a sufficient amount of arsenic trioxide todeliver a dose in the range of 5 to 10 mg.
 12. The unit dosage form ofclaim 11, further comprising one or more pharmaceutically acceptableexcipients.
 13. The unit dosage form of claim 11, wherein the unitdosage form is selected from the group consisting of solutions,suspensions, emulsions, syrups, tablets, and capsules.